We have shown that the Runx2 C terminus is required for bone development in vivo. Two principal co regulatory proteins that interact with the C terminus are the SMADs which mediate TGFP and BMP responsiveness and YAP (Yes associated protein) which mediates Yes/Src non receptor tyrosine kinase signaling. These factors interact with the Runx2 at a segment which overlaps the amino acid sequence essential for targeting Runx2 to its unique subnuclear domain, suggesting that interaction of Runx2 with BMP responsive SMADs and Yes/Src responsive YAP in subnuclear foci is essential for cellular differentiation in bone. We recently provided evidence that Runx2 is present in the multinucleated osteoclast and its mononuclear precursor. We postulate that the bone related Ruril transcription factor recruits its co regulatory proteins to specialized subnuclear domains and thal this nuclear organization of Runx SMAD and Runx YAP complexes with associated corepressor anc coactivator proteins is a requirement for Runx2 to function in regulating expression of numerous geneE involved in bone cell functions. This project focuses on functions of the SMAD and YAP co regulatory proteins ir regulating Runx2 transcription and their requirement to be at Runx sites in the nucleus to integrate Runx dependent osteoblast and osteoclastic signals. The four specific aims will 1) characterize the mechanism of Runx SMAC complex formation in subnuclear domains and the functional activity of this association as a requirement for osteoblast differentiation in vitro, 2) determine in vivo consequences of disruption of the Runx2 SMAD interactions by a gene replacement in the mouse that introduces a point mutation abrogating the Runx SMAD interaction, 3) define functional activity of Runx YAP complexes within the context of nuclear architecture as a transcriptional mediator of YES/Src signaling in osteoblasts, and 4) identify the role of Runx and Runx YAP complexes in the regulation of osteoclast differentiation and activation within the context of nuclear organization. Recruitment of these co regulatory proteins to Runx subnuclear domains provides a novel mechanism for regulating Runx mediated gene expression. These studies, characterizing the organization and function of Runx2 with its co regulatory proteins in subnuclear domains, will provide innovative insights for understanding mechanisms for cell type specific gene expression and bone pathologies related to Runx control of bone formation and resorption.